The CLARITY, a method of tissue clearing, was developed by Karl Deisseroth et al. at the Stanford University of the US in 2013, and published in “Nature” (Chung K et al., Nature, 497: 332-337 (2013)).
Specifically, according to this method, certain components within a biological tissue are replaced with an exogenous material of hydrogel, which results in transparent tissue and accordingly allows it to be optically detected. That is, this method is to visualize brain tissue by separating lipid components in the brain through a chemical treatment process.
In accordance with the conventional anatomical methods for analyzing microscopic structure and neuronal networks in a brain, the brain tissue is cut into thin slices to understand the respective neuronal networks thereof first, and followed by the integration of the large amount of information obtained from the slices to analyze overall structure of the brain cells and molecular distribution.
The CLARITY method of Karl Deisseroth et al., however, is worthwhile in that it makes it possible to detect neuronal networks using a transparent brain tissue in macroscopic or molecular level without impairing the tissue, unlike the conventional methods. Therefore, the CLARITY is expected to highly contribute in the brain disease research and biological tissue assays, etc.
FIG. 1, which is included in the Nature above, illustrates the CLARITY method and briefly explains a device for clearing lipid components from mouse brain tissue. In accordance with the FIG. 1, the brain of a mouse is contained in a chamber at first, and platinum wires are placed on opposite sides of the tissue, and then 10˜60 V electricity is applied thereto to separate the lipid components from the tissue by electrophoresis. While the negative charges (anions) (−) generated from the cathode move to the anode during the electrophoresis, ionized micelles surrounding the lipid components separate the lipid from the brain and keep on moving to the anode. Meanwhile, the chamber has a pair of inlet port and outlet for circulating the buffer solution which is circulated by a water circulator. Such a circulation is carried out for the secure of buffering capacity and replacing contaminated buffer solution. In addition, the circulation of buffer solution is used to reduce temperature in the chamber when the temperature therein is elevated by the electricity application.
However, the CLARITY method has a few drawbacks as follows: i) The surface area of the electrodes is small but the resistance is high compared to the plate electrodes since it employs wire electrodes, and thereby relatively higher electric voltage is applied to the electrodes at the same electric current. Further, the higher voltage results in high temperature heat generation, which increases the amount of side reactions, such as, electrolysis of water or organic materials, and accelerates contamination rate of the solution. ii) It is difficult to control heat generation since it is not easy to cool down the wire electrodes comparing to the plate electrodes. iii) The buffer solution circulates as rapidly as several litters per minute, so that it increases the amount of the side reactions. iv) It takes from 5 days to 9 days to separate lipid components from mouse brain using the CLARITY method.
In this regard, it is still required to develop a novel tissue clearing method which can remove or separate lipid components or constituents form a biological tissue more rapidly, while controlling heat generation and decreasing circulation rate of buffer solution to reduce the amount of side reactions.